The medical use of fullerene derivatives is based on the lipophilic properties of the fullerene core, which enables fullerene derivatives to permeate cellular membranes, and the ability of fullerenes to generate in high quantum yield singlet oxygen, which splits DNAs. These properties endow functional fullerene derivatives with cytotoxic, antiviral, and other properties (see Bedrov, D., Smith, G. D., Davande, H., “Passive transport of fullerenes through a lipid membrane,” J. Phys. Chem., B, 2008, Vol. 1 12., pp. 2078-84; Qiao, R., and Roberts A. E., “Translocation of fullerene and its derivatives across a lipid bilayer”, Nano Lett., 2007, Vol. 7, pp. 614-9; Nelsen, G. D., et al., “In vivo biology and toxicology of fullerenes and their derivatives”, Basic and Clinical Pharmacology and Toxicology, 2008, Vol. 103, pp. 197-208; and U.S. Pat. No. 6,204,391, 2005, “Water soluble fullerenes with antiviral activity”).
The main problem hampering biological studies of fullerene derivatives and the creation of medicaments on their basis arises from the water insolubility of fullerenes, which hampers their direct administration into a human body. One possible way to overcome these difficulties is to embed fullerene molecules into solubilizing matrices. Methods are known for preparing water-soluble fullerene species through forming a polyvinylpyrrolidone adduct (see Kiselev, O. I., et al., Mol. Materials, 1998, Vol. 11, p. 121; Piotrovsky, L. B., et al., Ibid., 2000, Vol. 13, p. 41). This adduct was shown to be efficient against influenza A and B viruses.
Further, a method is known for preparing fullerenes, which comprises mixing fullerenes pre-dissolved in an organic solvent with a polymeric matrix in chloroform, concentrating the mixture under vacuum until the solvents are completely removed, and dissolving the resulting complex in a phosphate salt buffer (pH 7.4-7.6), followed by an ultrasonic treatment of the product (see RU No. 2162819. Oct. 2, 2001). The water-soluble polymeric matrices used according to that patent are membrane cephalins. The products obtained as a result of such modifications are unstable compositions having limited storage potentials.
A promising method for preparing water-soluble fullerene compositions is offered by chemical modification of the fullerene sphere by incorporation of hydrophilic solubilizing ligands. The international application WO2005/070827 shows a set of amino acid derivatives of fullerene prepared through the cycloaddition reaction of amino acid moieties to fullerene, and the products of their insertion into biologically active organic substrates. The synthesis methods disclosed in that technical solution are multistep and poorly adaptable. The resulting compounds have low water solubilities.
Currently, a wide range of functionalized fullerenes have been prepared, wherein hydrophilic moieties are present in the side chains of ligands attached to the fullerene (the detergent type of complex), as well as spherical derivatives wherein polar groups are distributed over the fullerene sphere (this type includes fullerenols and amino adducts).
Amino acid derivatives of fullerenes have the greatest potential for use.
Analogues of the present invention are the compounds and methods for producing them as described in the international application WO2009/00203 and those described in the Russian Federation patent No. 2236852.
The international application WO2009/00203 described polyfunctional amino acid derivatives of formula

where R═H, mono- or dihydroxyalkyl, haloalkyl, mono- or dinitroxyalkyl, maleinimde; N—Z is a moiety of an α, β, γ, or ω amino acid of general formula —NH—CmH2m—COOM or C4H8N—COOM, where m=2-5, and M is a nitroxyalkyl group, alkyl group, or an alkali metal salt, or a dipeptide. These compounds were prepared via the reaction of equimolar addition of an amino acid to fullerene, followed by the substitution of a biologically active organic ligand for hydrogen to form the type of compound. The resulting compounds have an inhibitory activity against metastasizing tumors, enhance the antileukemic activity of cyclophosphamide, and can be suitable as nitrogen monoxide donors or as fast acting vasodilators for antihypertensive therapy.
The major drawback of the compounds according to that application consists in that they are covalent addition products, contain small amounts of polar groups, and have low water solubilities.
The most pertinent piece of prior art in the context of the technical essence and the attainable result consists of an agent for inhibiting the reproduction of enveloped viruses and the method of preparing same according to RU patent No. 2236852. As a result of reacting a fullerene with an amino acid salt in an organic solvent in the presence of polyalkylene oxide, fullerene-polycarboxylic anions of general formula C60Hn[NH(CH2)mC(O)O−]n were prepared, wherein C60 is the fullerene core, NH(CH2)mC(O)O− is an aminocarboxylic anion; m is an integer from 1 to 5, and n is an integer from 2 to 12.
In order to prepare these compounds, to a solution of fullerene in o-dichlorobenzene (or toluene or another organic solvent), an amino acid is inserted as a salt (potassium or sodium salt) and then a solubilizing agent is added. The order of addition of the amino acid and solubilizing agent is unimportant; they can be added as a premixed complex. Useful solubilizing agents are various polyalkylene oxides (polyethylene glycols having molar weights from 150 to 400 or higher than 400 (for example, PEG-1500), as well as polyethylene glycol dimethyl ether having a molar weight of 500. In order to increase reaction rates, any strong reducing agent (an alkali metal) is added.
The fullerene-to-amino acid ratio is increased by more than 50 times. Conversion to the desired pharmaceutically acceptable salt, especially, a sodium or potassium salt, was performed by treating the acid with a suitable base or by adding a salt of a weak volatile acid. In particular, a water-insoluble fullerene-polycarboxylic acid is converted to a more preferable pharmaceutically acceptable, water soluble salt, for example to a sodium salt. Addition of a salt of a weak volatile acid is performed via treating the solution with an alkali metal salt of a weak volatile acid. Upon concentrating the solution by evaporation or freeze drying, the weak acid is removed and fullerene-polycarboxylic acids are recovered as their alkali metal salts. The target product of the invention has a constant composition; the content of the major substance in the target product is as low as 87.8%. The specification lacks flowsheet protocols for the determination of optimal amounts of the starting compounds, the ratios of amounts of the solvents used, and most important, the description of methods for isolating the sought compounds.
The major drawbacks of the fullerene amino acid derivatives prepared by the method shown in the cited patent consist of producing a mixture of fullerene-carboxylate anions in the form of both salt and acid species. An individual compound cannot be prepared by the method described in the cited patent. Furthermore, the fullerene poly(amino acids) prepared by this prior-art method in the acid form are almost water insoluble. Attempts at preparing a stable pharmaceutical composition with fullerene-polycarboxylic anions failed, because compounds are precipitating during storage.
The necessity of using in the synthesis of great excesses of a potassium or sodium salt of amino acids and great excesses of solvents gives rise to environmental problems in waste recycling, and increases the cost of the production process. For technological reasons alkali metals cannot be used to increase the reaction rate when chlorinated aromatic solvents are used.
However, the unique biochemical properties of agents comprising fullerene compounds with amino acid moieties described in the cited patent pose the problem of preparing new fullerene derivatives, developing a highly adaptable large-scale process for their production that would be distinguished by simplicity and efficiency, freedom from contaminants, environmental safety, and availability of the starting reagents.